Group C streptococci are a common cause of infection in several animal species but are generally considered to be a rare cause of infection in humans (Ghoneim, A. T. et al. (1980), J. Clin. Pathol. 33:188-190; Feldman, W. E. Postgrad. Med. (1993), 93(3): 141-145). Of the four species of group C streptococci, S. equisimilis has been reported to cause most human illnesses, including bacteremia, endocarditis, meningitis, pneumonia, epiglottitis, puerperal sepsis, and wound infections. However, S. zooepidemicus has been associated with two outbreaks of pharyngitis and nephritis in Europe (Duca, E. et al. (1969), J. Hyg. Camb. 67: 691-698; Barnham, M. et al. (1983), Lancet 8331: 945-948). In both of the European outbreaks, unpasteurized milk was suspected as the source of infection. More recently, Group C streptococci was identified as a causative agent in a prosthetic joint infection (Kleshinski, J. et al. (2000) Southern Medical Journal 93:1217-1220). This is the first reported outbreak in the United States and is the first such reported outbreak in which the vehicle, ie. cheese made from unpasteurized cows' milk, has been epidemiologically implicated. Although S. zooepidemicus and S. equisimilis are rarely reported causes of mastitis in cows, the cause of this outbreak was contaminated milk from cows with mammary infections due to S. zooepidemicus. 
Group C streptococci are also the causative agent of strangles, a highly contagious and serious infection of horses and other equids. The disease is characterized by severe inflammation of the mucosa of the head and throat, with extensive swelling and often rupture of the lymph nodes, which produces large amounts of thick, creamy pus. The organism can be isolated from the nose or lymph nodes of affected animals, and is usually readily identified in the laboratory by simple sugar tests.
Control of the disease is complicated by the development of long-term carriers that outwardly appear healthy and are frequently difficult to detect because swab samples from them often do not yield cultures of S. equi. The organism resides in the guttural pouches and resumption of active shedding can recur sporadically for unknown reasons. Previously, endoscopy was the only way to reliably detect most carriers but this is too impractical in most circumstances. The outbreak prevalence of these carriers combined with movement and mixing with susceptible animals probably accounts for the high incidence of strangles. Although a “PCR test” to detect the DNA of the organism in nasopharyngeal swabs is now available, enabling sensitive carrier detection, there are still practical difficulties and expense with multiple nasal sampling and some endoscopy, although much less than previously. For the large majority of horses the most effective means of controlling strangles would be a good vaccine or other immunogenic compositions that are effective in eliminating the causative agent.
Group A Streptococcus (Streptococcus pyogenes), the primary etiologic agent of bacterial pharyngitis, is one of few human pathogens that remain uniformly sensitive to penicillin (Macris, M. H., et al. (1998) Pediatr. Infect. Dis. J. 17:377-381). Additionally, the advent of rapid group A streptococcal diagnostic test kits over the last decade has allowed early initiation of antibiotic treatment. Despite these factors, streptococcal-mediated pharyngitis is reported in over 2.5 million people annually in the United States with >80% of these cases occurring in children under 15 years of age (Schappert, S. M., et al. (1999) Vital Health Stat. 13). However, streptococcal pharyngitis classically is not a reportable disease and it has been speculated that the documented number of these pharyngitis cases may be considerably underestimated. Additionally, penicillin fails to completely eradicate streptococci in up to 35% of patients treated for pharyngitis (Pichichero, M. E. (1998) Pediatr. Rev. 19:291-302) and carriage rates as high as 50% have been reported in close contact areas such as day care centers (Feldman, S., et al. (1987) J. Pediatr. 110:783-787). This high carriage rate contributes to the spread of streptococcal pharyngitis (Nguyen, L., et al. (1997) J. Clin. Microbiol. 35:2111-2114) and correlates with outbreaks of rheumatic fever (Oliver, C. (2000) J. Antimicrob. Chemother. 45 Topic T1:13-21). While eradication of the carrier state would reduce the pool of streptococci in the population, and thus streptococcal-related diseases, to date the only treatment is an extensive regimen of antibiotics (Tanz, R. R., et al. (1998) Pediatric Annals 27:281-285) that may increase streptococcal resistance to macrolides, which are often prescribed for patients with penicillin allergies (York, M. K., et al. (1999) J. Clin. Microbiol. 37:1727-1731).
Bovine mastitis is an inflammation of a cow's mammary gland, usually due to a microbial infection originating from contaminated teats. Experimental bovine mastitis can be induced with as little as 100 organisms, so a few chronic infections within a herd can maintain a persistent bacterial reservoir. Several bacterial species have the ability to cause bovine mastitis, including Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae (Group B strep), and Escherichia coli. Of these, S. aureus, which causes acute conditions, and S. uberis, which causes chronic conditions, are responsible for the bulk of bovine mastitis cases. The persistence and economic impact of bovine mastitis is alarming. Wilson et al. (Wilson, D. J., et al. (1997) J. Dairy Sci. 80:2592-2598) recently published the results of a retrospective study of milk samples collected from more than 100,000 cows in New York and northern Pennsylvania between 1991 and 1995. They found that intramammary infections were present in 36% of cows enrolled in the Dairy Herd Improvement Association. This disease is estimated to cost the producer approximately $200/cow/year, which corresponds to a U.S. total of $1.7 billion annually.
Current therapies for bovine mastitis rely heavily on the use of β-lactam antibiotics such as penicillins and cephalosporins. These agents have had a beneficial impact on dairy-animal health and milk production. However, the cure rate for treatment of some infections, particularly S. aureus, is often less than 15%. This is attributed to incomplete penetration of the antibiotics throughout the mammary gland (Yancey, R. J., et al. (1991) Eur. J. Clin. Microbiol. Infec. Dis. 10:107-113). Additionally, concerns of accidental exposure of susceptible consumers to residual antibiotics resulting in anaphylaxis has necessitated the imposition of a post-treatment milk discard period and strict industry surveillance of all milk shipments. While infections can be cleared in days with antibiotic treatment, the discard period can often last weeks until residual antibiotic levels fall within acceptable parameters. Finally, there is growing concern that the agricultural use of antibiotics contributes to the emergence of antibiotic resistance in human pathogens (Smith, D. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:6434-5439). Taken together, these concerns suggest alternative therapies are needed for the therapeutic management of bovine mastitis.
Tailed bacteriophages are the most populous “organism” on Earth with roughly 1030 inhabitants in the biosphere (Brussow, H. et al. (2002) Cell 108:13-16). However, we are just beginning to appreciate the role they play in bacterial diversity (Hendrix, R. W. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2192-2197) and more recently, bacterial pathogenesis (Broudy, T. B. et al. (2003) Infect. Immun. 71:3782-3786; Wagner, P. L. et al. (2002) Infect. Immun. 70:3985-3993). Indeed, whole genome sequencing of two different strains of group A streptococci reveals that polylysogenic phage represent the only diversity between the two strains (Beres, S. B. et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:10078-10083; Ferretti, J. J. et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663). Recent advances have allowed whole bacteriophage genomes of evolutionary or biological interest to be rapidly sequenced for comparison to known genomes.
The streptococcal C1 bacteriophage has roots at the forefront of bacteriophage research. Shortly after the discovery of bacteriophages by Twort and d'Herelle (Twort, F. W. (1915) Lancet ii.: 1241-1243; d'Herelle, F. H. (1917) C. R. Acad. Sci. (Paris) 165:373-375), the C1 bacteriophage was isolated by Clark in 1925 from a sewage plant in Milwaukee, Wis. and represents the first documented bacteriophage found to be active on any type of streptococci (Clark, P. F. et al. (1926) J. Bacteriol. 11:89). Initially known as the “sludge” phage or “Clark” phage, it infected streptococci isolated from animals (which we now know to be group C streptococci), but not streptococci found in humans (now known to be group A streptococci) (Shwartzman, G. (1927) J. Exp. Med. 46-497-509; Lancefield, R. C. (1932) Proc. Soc. Exp. Biol. Med. 30:169-171). In a hallmark 1934 paper, Alice Evans, using the “Clark” phage which she renamed B563, was the first person to utilize phage in order to classify bacterial strains, thus founding the analytical field of phage typing (Evans, A. C. (1934) Public Health Reports 49:1386-1401). Additionally, Evans noticed that phage lysates had lytic activity on streptococci that were not sensitive to the phage itself. She called this phenomenon “nascent lysis” and attributed it to a “lysin” activity originally defined by Twort (Twort, F. W. (1925) Lancet ii.:642-644). In 1957, Krause renamed the Evans B563 phage, C1, to imply an exquisite specificity for group C streptococci (Krause, R. M. (1957) J. Exp. Med. 106:365-384). Krause also noted that the C1 cell wall hydrolase, or lysin, had a less restrictive range given that groups A, C, and E streptococci were rapidly lysed by this enzyme.
On the genetic level, the C1 phage has not been studied in detail. Two published restriction maps of this genome exist (Pomrenke, M. E. et al. (1989) J. Basic Microbiol. 6:395-398; Totolian, A. A. et al. (1981) Reedbooks Ltd., Surrey), but to date, no sequence data was available. The majority of interest in this phage involves its lysin, which has been used extensively as a tool to dissolve the streptococcal cell wall in order to make protoplasts, extract genomic DNA, or to study surface proteins (van de Rijn, I. et al. (1981) Infect. Immun. 32:86-91; Wheeler, J. et al. (1980) J. Gen. Microbiol. 120:27-33).
At the end of a bacteriophage lytic cycle in a sensitive bacterial host, all double stranded DNA bacteriophages produce a lytic system that consists of a holin and at least one peptidoglycan hydrolase, or “lysin”, capable of degrading the bacterial cell wall. Lysins can be endo-β-N-acetylglucosaminidases or N-acetylmuramidases (lysozymes), which act on the sugar moiety, endopeptidases which cleave the peptide cross bridge, or more commonly, an N-acetylmuramoyl-L-alanine amidase, which hydrolyzes the amide bond connecting the sugar and peptide constituents. Typically, the holin is expressed in the late stages of phage infection forming a pore in the cell membrane allowing the lysin(s) to gain access to the cell wall peptidoglycan resulting in release of progeny phage (for review, see (Young, R. (1992) Microbiol. Rev. 56:430-481)). Lysin, added to sensitive organisms in the absence of bacteriophage, lyses the cell wall producing a phenomenon known as “lysis from without”.
The C1 bacteriophage specifically infect group C streptococci and produce a lysin (termed PlyC) that has been partially purified and characterized (Fischetti, V. A., et al. (1971) J. Exp. Med. 133:1105-1117; Raina, J. L. (1981) J. Bacteriol. 145:661-663). C1 phage lysin can cause “lysis from without” in groups A and E streptococci as well as group C streptococci (Maxted, W. R. (1957) J. Gen. Microbiol. 16:584-594; Krause, R. M. (1957) J. Exp. Med. 106:365-384). Additionally, PlyC works on Streptococcus equi and Streptococcus uberis. This unique activity has been exploited as a tool in group A streptococcal studies to isolate surface molecules including M proteins (Fischetti, V. A., et al. (1985) J. Exp. Med. 161:1384-1401), to lyse cells for DNA extraction, and to make protoplasts when used in a hypertonic environment (Wheeler, J., et al. (1980) J. Gen. Microbiol. 120:27-33).
The present invention provides alternate means for the prevention and/or treatment of pathogenic streptococcal infections in humans and animals such as streptococcal pharyngitis, equine Strangles disease, bovine mastitis, and other disease states associated with groups A, C, and E streptococci as well as S. equi and S. uberis. Furthermore, the present invention provides the means for diagnosis of such pathogenic infections.